Analysis of the Function of Apoptosis during Imaginal Wing Disc Regeneration in <i>Drosophila melanogaster</i>

<div><p>Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of <i>Drosophila melanogaster</i> provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, we have defined the pattern of cell death in regenerating discs. Our data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. We also present results which suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, our results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.</p></div

Regenerative growth is reduced in <i>egr</i>^<i>1</i><i>/egr</i>^<i>3</i> regenerating discs.

<p>(A-B’) Third instar wing discs stained for the mitotic marker phospho-Histone H3 (blue in A-B, and grey in A’-B’) and anti-Wg (red in A-B). (A-A’) Control <i>HhdsRed Ci-Gal4 UAS-GFP/+</i> disc. (B-B’) Regenerating <i>eiger</i>^<i>3</i><i>/eiger</i>^<i>1</i>; <i>HhdsRed Ci-Gal4 UAS-GFP/+</i> discs at 20 hrs after cut. We found that there is a reduction in the number of mitotic cells in both compartments. (C) Bar charts show the average fold change in the mitotic index of control regenerating discs (control), and <i>eiger</i>^<i>3</i><i>/eiger</i>^<i>1</i>;<i>HhdsRedCi-Gal4 UAS-GFP/+</i> regenerating discs (<i>eiger</i>^<i>3</i><i>/eiger</i>^<i>1</i>) at 20 hrs AC, compared to control non-regenerating discs.</p

Apoptosis is reduced in <i>egr</i>^<i>1</i><i>/egr</i>^<i>3</i> regenerating discs.

<p>(A-B’) Third instar wing discs stained for the apoptotic marker anti-cleaved Caspase-3 (blue in A-B, and grey in A’-B’). (A-A”) Control <i>HhdsRed-CiGal4 UAS-GFP/+</i> regenerating discs amputated in the anterior compartment. (B-B”) Regenerating <i>eiger</i>^<i>3</i><i>/eiger</i>^<i>1</i>; <i>HhdsRed Ci-Gal4 UAS-GFP/+</i> discs at 20 hrs after cut. We found that there is a reduction in the number of dead cells in both compartments. (C) Bar chart shows the average apoptotic index in control regenerating discs amputated in the anterior compartment (Control) and <i>eiger</i>^<i>3</i><i>/eiger</i>^<i>1</i>; <i>HhdsRed Ci-Gal4 UAS-GFP/+</i> (<i>eiger</i>^<i>3</i><i>/eiger</i>^<i>1</i>) regenerating discs at 20 hrs AC. Note that the apoptotic index of discs amputated in the anterior compartment is very similar to that observed in discs amputated in the posterior compartment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165554#pone.0165554.g002" target="_blank">Fig 2</a>). Schematic illustrations on the left indicate the cutting lines and the regions eliminated in each disc.</p

Ectopic activation of JNK signalling is not sufficient to induce the”non autonomous” cell death observed in regenerating control discs.

<p>(A-C’) Third instar <i>en</i>-<i>Gal4 UAS-eiger UAS-GFP /Tub-Gal80</i>^<i>ts</i> wing discs (A-A’), and <i>en</i>-<i>Gal4 UAS-hep</i>^<i>CA</i> <i>UAS-GFP /Tub-Gal80</i>^<i>ts</i> wing discs (B-C’). Larvae were shifted from 17°C to 29°C for 24 hrs before the staining. The discs were stained with anti-cleaved Caspase-3 (red in A-C, and grey in A’-C’); anti-Wg (blue A-B). (A-A’) The ectopic expression of <i>eiger</i> in the posterior compartment only induces the apoptosis of a few scattered cells in the anterior compartment (arrows), compared with regenerating discs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165554#pone.0165554.g001" target="_blank">Fig 1</a>. (B-B’) Although cell death is massively induced in the posterior compartment of <i>en</i>-<i>Gal4 UAS-hep</i>^<i>CA</i> <i>UAS-GFP /Tub-Gal80</i>^<i>ts</i> discs, we only found a few apoptotic cells in the anterior compartment (arrows). (C-C’) High magnification of <i>en</i>-<i>Gal4 UAS-hep</i>^<i>CA</i> <i>UAS-GFP /Tub-Gal80</i>^<i>ts</i> discs. Most apoptotic cells in the A/P boundary are posterior cells, since they also express GFP (arrowheads), only a few dead cells are anterior (arrows). (D) Bar chart shows the average apoptotic index in the anterior compartment of control regenerating discs (control), <i>en</i>-<i>Gal4 UAS-eiger UAS-GFP /Tub-Gal80</i>^<i>ts</i> (enG4 eiger) and discs <i>en</i>-<i>Gal4 UAS-hep</i>^<i>CA</i> <i>UAS-GFP /Tub-Gal80</i>^<i>ts</i> (enG4 hepCA). Cell death index 0,000251323±6,98223E-05, n = 5 and 0,00027647 ± 3,84519E-05, n = 5 in the anterior compartment of discs over-expressing <i>hep</i>^<i>CA</i> and <i>eiger</i> respectively vs 0,00119 ±0,00042,n = 8 in the anterior compartment of control regenerating discs.</p

Expression of Wg and Delta in regenerating <i>en-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+</i> discs 20 hrs AC.

<p>(A-B’) Expression of Wg, shown by anti-Wg (red in A-B, and grey A’-B’). (C-D’) Expression of Delta (red in C-D, and grey C’-D’). (A-A’ and C-C’) Control <i>en-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+</i> third instar non amputated discs. (B-B’ and D-D’) <i>en-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+</i> regenerating discs at 20hrs AC. On the left, illustrations of the discs are shown indicating the cutting lines and the regions eliminated in the discs shown by panels A-D. (B-B’) Example of an <i>en-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+</i> regenerating disc at 20 hrs AC in which the expression of Wg disappears in the d/v boundary near the wound edge (arrowhead in B’). In this disc the expression of the internal ring of Wg was restored. (D-D’) The vein/intervein pattern defined by Dl was disrupted in <i>en-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+</i> regenerating discs dissected 20 hrs AC (D-D’ compared to C-C’).</p

The over-expression of <i>dIAP</i> is not sufficient to block apoptosis during disc regeneration.

<p>(A-D”) Third instar wing <i>en-Gal4 UAS-GFP</i> / <i>nub-Gal4; UAS-dIAP1</i>/+ discs stained for anti-cleaved Caspase-3 (blue in A-D, and grey in A”-D”) and anti-Wg (red A-D and grey A’-D’). (A-A”) Control <i>en-Gal4 UAS-GFP</i>/<i>nub-Gal4; UAS-dIAP1</i>/+ disc. (B-B”) Regenerating <i>en-Gal4 UAS-GFP</i> / <i>nub-Gal4; UAS-dIAP1</i>/+ discs at 3 hrs AC. As in control discs, we found a few dead cells at the wound edge or in the region adjacent. (C-C”) 20 hrs AC, we observed a high number of dead cells throughout the wing blade. (D-D”) 48 hrs AC, as observed in control discs, at this time there is a reduction in the number of dead cells in both compartments. (E) Bar chart shows the average apoptotic index in control regenerating discs (Control) and <i>en-Gal4 UAS-GFP</i> / <i>nub-Gal4; UAS-dIAP1</i>/+ (<i>nubG4 enG4 UAS dIAP1</i>) discs at 20hrs AC. Here and in the rest of figures the error bars represent the standard deviation. Schematic illustrations on the left indicate the cutting lines and the regions eliminated in each disc.</p

Characteristics of the individuals with acquired brain injury.

Characteristics of the individuals with acquired brain injury.</p

Characteristics of clinicians.

The objective was to explore the care experiences and service design related to rehabilitation for mobility and participation in the community among individuals with acquired brain injury (ABI), as perceived by clinicians and patients. Five focus groups were held: three with clinicians and two with individuals with ABI. Focus group discussions were transcribed and analyzed using an inductive and deductive thematic content approach. Five themes were identified: Enabling continuity of care; System design; Accessibility and services in the community; Transportation services; and Uncertainty about the provided services. The results of participants’ experiences contributed to developing recommendations of service provision for mobility, leading to a patient-centered continuum of rehabilitation services. Accessibility to rehabilitation to improve the quality of care by addressing needs during transitions and mobility-related deficits, providing needed information, coordinated care, and self-management support in the community.</div

Additional file 1: of Age, gender, and current living status were associated with perceived access to treatment among Canadians using a cross sectional survey

This file provides additional data tables describing our study population and analysis. Appendix 1 provides a brief overview of how we constructed the summary variables for several multi-question concepts, including perceived affordability of the healthcare system or its services and medication adherence. Appendix 2 provides a frequency distribution of the CHCs reported by patients in the study population. Appendix 3 provides a frequency distribution for the additional covariates included in the ordinal regression model, outside of the sociodemographic factors seen in Table 1. These frequencies are stratified by the respondents’ reported level of access to treatment(s) needed to manage their CHCs. Appendix 4 provides the results from the univariate regression analysis. Appendix 5 looks into the self-reported reasons for not always having access to treatment to manage CHCs, cited by patients in the study population who perceive their level of access as “Often” or “Sometimes/Rarely/Never.”. (DOCX 84 kb

Inductive and deductive thematic analysis.

S3 Table legend C: clinician perspective; S: stroke perspective; T: traumatic brain injury perspective. (PDF)</p